Journal of Signal Transduction / 2015 / Article / Fig 2

Research Article

Analysis of AKAP7 Dimerization

Figure 2

AKAP7γ forms homooligomers displaying high affinity interactions. (a) Lysates from AKAP7γ-EGFP transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7α (lanes 2 and 3) or AKAP7γ (lanes 4 and 5) precharged on S-protein resin. Anti-GFP antibody was used for detecting protein interactions by western blot analysis (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μL) is shown in lane 1; . (b) Lysates from AKAP7α-EGFP transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7α (lanes 2 and 3) or AKAP7γ (lanes 4 and 5) precharged on S-protein resin. Anti-GFP antibody was used for detecting protein interactions by western blot analysis (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μL) is shown in lane 1; . (c) SPR was performed by immobilizing AKAP7γ (150 RU, black) and AKAP7α (150 RU, red) in separate flow chambers on a CM5 chip and measuring the response when passing over a range of concentrations (25–200 nM) of either AKAP7γ (c) or AKAP7α (d). The sensorgram of AKAP7γ analyte binding to AKAP7γ ligand (c, black lines) was best fit with a heterogeneous analyte model suggesting the presence of two binding sites on AKAP7γ. The affinities of the two binding sites, as determined by the Biacore T100 evaluation software, were 0.537 nM and 79.7 nM with forward () and reverse rates () of association as shown. (e) Lysates from AKAP7δ-EGFP transfected HEK-293 cells were subjected to pulldown assays using bacterially purified S-tagged AKAP7γ (lane 1) or AKAP7α (lane 2) precharged on S-protein resin. Anti-YFP antibody was used for detecting protein interactions by western blot analysis (upper panel). Protein stain of the nitrocellulose membrane before western blot analysis is shown in the lower panel. Input from the transfected cells (20 μL) is shown in middle panel; . (f) Western blot analysis of anti-mCherry immunoprecipitates isolated from HEK-293 cells cotransfected with AKAP7δ-YFP in the absence (lane 1) and presence (lane 2) of AKAP7γ-mCherry (upper panel). To confirm immunoprecipitation of AKAP7γ-mCherry, the nitrocellulose membranes were stripped and reprobed for mCherry using a goat polyclonal anti-DsRed antibody (middle panel). Lower panel depicts mCherry western blot analysis of the input from each condition; .
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